This project has been concerned with study of the composition, structure, mechanism of action and interaction of mitochondrial enzyme complexes I (NADH:ubiquinone, Q, oxidoreductase), II (succinate: Q oxidoreductase), III (ubiquinol: cytochrome c oxidoreductase), and V (ATP synthase). The studies proposed here include the following: Complex I - (a) Continuation of the use of controlled and differential proteolysis to identify the electron paramagnetic resonance-visible iron-sulfur clusters associated with the 75 and the 23 kDa subunits, and their involvement in electron transfer from NADH to Q. (b) We have discovered that bovine complex I contains 4 nicotinamide nucleotide- binding subunits not involved in electron transfer from NADH to Q. These are subunits 42 kDa, NAD(H)-specific; 39 kDa, NADP(H)-specific; 30 kDa, NAD(H)/NADP(H)-binding; and 18-20 kDa, NADP(H)-specific. The possible role of these subunits together with the acyl carrier protein of complex I in de novo intramitochondrial fatty acid synthesis will be investigated. Complex III - Continuation of the study of the mechanism of electron transfer by complex III, using Rhodobacter capsulatus chromatophores containing mutations in the heme bL domain of cytochrome b or in the extrinsic domain of the iron-sulfur protein (ISP). These studies are important, because (a) our results with the bovine system have indicated that Q is not necessary for electron transfer between bL and ISP, (b) complex III x-ray diffraction results of two groups do not show Q in this region, and (c) the extrinsic domain of ISP appears to swing between cytochromes b and c1. Complex V - (a) Study of the involvement of subunits b, d, e, f g, and A6L as possible membrane-bound anchors for the ATPase inhibitor protein and factor B. (b) Study of the possible involvement of subunits F6, d, e, f g and A6L together with subunits b and OSCP as components of the stator of the bovine mitochondrial ATP synthase. Mitochondrial defects cause a variety of debilitating human neuromuscular degenerative diseases, whose prevention and treatment require a knowledge of the structure and function of mitochondria at the molecular level.